TY - JOUR
T1 - Real-Time Visualization of in Vitro Transcription of a Fluorescent RNA Aptamer
T2 - An Experiment for the Upper-Division Undergraduate or First-Year Graduate Laboratory
AU - Heili, Joseph M.
AU - Gomez-Garcia, Jose
AU - Gaut, Nathaniel J.
AU - Cash, Brock W.
AU - Aufdembrink, Lauren M.
AU - Heffron, Brent A.
AU - Shirley, Joshua D.
AU - Carlson, Erin E.
AU - Adamala, Katarzyna P.
AU - Engelhart, Aaron E.
N1 - Publisher Copyright:
© 2018 American Chemical Society and Division of Chemical Education, Inc.
PY - 2018/10/9
Y1 - 2018/10/9
N2 - A teaching laboratory experiment is described where students prepare in vitro transcription reactions of a fluorescent RNA aptamer, named Broccoli, and observe the production of the aptamer in real-time on a fluorescence plate reader. Alternate visualization methods with minimal costs are also described for laboratories lacking this instrumentation. Two optional experiments are also described. Optional Experiment 1 involves purification of RNA transcription reactions using a commercial spin column kit and having students correlate cleanup kit yield with transcribed aptamer fluorescence. Optional Experiment 2 involves running a polyacrylamide gel of the transcription reaction with a ladder, followed by staining with (Z)-4-(3′,5′-difluoro-4′-hydroxybenzylidene)-2-methyl-1-(2″,2″,2″-trifluoroethyl)-1H-imidazol-5-(4H)-one (DFHBI-1T) (selective for Broccoli) and a second stain with SYBR Gold (nonselective, allowing for simultaneous visualization of Broccoli and ladder). This experiment has the practical advantage of enabling aptamer visualization in laboratories without a fluorescence spectrometer or plate reader, as well as the pedagogical benefit of demonstrating specific activation of the fluorescence of a small molecule by an RNA aptamer in another context (gel staining). Each experiment allows students to perform straightforward, easily understood teaching laboratory experiments, including key concepts in cellular imaging, and RNA biochemistry widely employed in biochemical research.
AB - A teaching laboratory experiment is described where students prepare in vitro transcription reactions of a fluorescent RNA aptamer, named Broccoli, and observe the production of the aptamer in real-time on a fluorescence plate reader. Alternate visualization methods with minimal costs are also described for laboratories lacking this instrumentation. Two optional experiments are also described. Optional Experiment 1 involves purification of RNA transcription reactions using a commercial spin column kit and having students correlate cleanup kit yield with transcribed aptamer fluorescence. Optional Experiment 2 involves running a polyacrylamide gel of the transcription reaction with a ladder, followed by staining with (Z)-4-(3′,5′-difluoro-4′-hydroxybenzylidene)-2-methyl-1-(2″,2″,2″-trifluoroethyl)-1H-imidazol-5-(4H)-one (DFHBI-1T) (selective for Broccoli) and a second stain with SYBR Gold (nonselective, allowing for simultaneous visualization of Broccoli and ladder). This experiment has the practical advantage of enabling aptamer visualization in laboratories without a fluorescence spectrometer or plate reader, as well as the pedagogical benefit of demonstrating specific activation of the fluorescence of a small molecule by an RNA aptamer in another context (gel staining). Each experiment allows students to perform straightforward, easily understood teaching laboratory experiments, including key concepts in cellular imaging, and RNA biochemistry widely employed in biochemical research.
KW - Biochemistry
KW - Electrophoresis
KW - Fluorescence Spectroscopy
KW - Graduate Education/Research
KW - Hands-On Learning/Manipulatives
KW - Nucleic Acids/DNA/RNA
KW - Upper-Division Undergraduate
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U2 - 10.1021/acs.jchemed.7b00735
DO - 10.1021/acs.jchemed.7b00735
M3 - Article
AN - SCOPUS:85054638314
SN - 0021-9584
VL - 95
SP - 1867
EP - 1871
JO - Journal of Chemical Education
JF - Journal of Chemical Education
IS - 10
ER -